title: Mass spectrometric analysis of the S-layer proteins from Clostridium difficile demonstrates the absence of glycosylation.
abstract: Like many other bacterial cell surfaces, the cell wall of Clostridium difficile is also encapsulated by a proteinaceous paracrystalline layer, the surface (S)-layer. In many bacterial species, the S-layer proteins (SLPs) have been shown to be glycosylated, whereas in other species glycosylation is absent. Unusually, the S-layer of C. difficile is composed of two distinct proteins, the high-molecular weight (HMW) and low-molecular-weight (LMW) SLPs. Previous investigations have reported that one or both of these SLPs are glycosylated, though no definitive study has been conducted. We have used a variety of mass spectrometric approaches to analyse SLPs from a number of strains of C. difficile for the presence of associated glycans. Analysis of intact SLPs by matrix assisted laser desorption/ionisation time of flight (MALDI-ToF) mass spectrometry demonstrated that the observed molecular masses matched the predicted masses of the LMW and HMW SLPs. Furthermore, analysis of Cyanogen bromide (CNBr) and tryptic peptides displayed no evidence of post-translational modification. In the first in-depth study of its kind, we unequivocally demonstrate that the S-layer proteins from the C. difficile strains investigated are not glycosylated.
authors.affiliation: Division of Molecular and Cell Biology, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
authors.forename: Omar
authors.initials: O
authors.lastname: Qazi
authors.forename: Paul
authors.initials: P
authors.lastname: Hitchen
authors.forename: Bérangère
authors.initials: B
authors.lastname: Tissot
authors.forename: Maria
authors.initials: M
authors.lastname: Panico
authors.forename: Howard R
authors.initials: HR
authors.lastname: Morris
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Neil
authors.initials: N
authors.lastname: Fairweather
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016360:Clostridioides difficile / Q000737:chemistry* / Q000235:genetics; D003488:Cyanogen Bromide / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C055764:S-layer proteins; D014357:Trypsin; D003488:Cyanogen Bromide